Method for designing probe, system for designing probe, and program for designing probe

Abstract

To provide a system for designing a probe capable of reducing false detection.SOLUTION: A system for designing a probe comprises: a measurement target sequence acquisition section 301 for acquiring the sequence of molecules to be measured; and a sequence unit generation section 302 for generating a sequence unit that satisfies a constraint condition where a k-mer spanning a linking point of a linked sequence formed by linking the sequence units is not complementary to a k-mer in which the frequency of occurrence of the molecules to be measured is greater than or equal to a predetermined value.SELECTED DRAWING: Figure 10

Description

[Technical field]

[0001] The present invention relates to biotechnology and to a probe design technique. [Background technology]

[0002] In recent years, the importance of transcriptome analysis has increased in understanding the relationship between gene mutations and complex diseases such as cancer, diabetes, and heart disease. Fluorescence In Situ Hybridization (FISH), one of the methods of transcriptome analysis, can detect the expression distribution and expression level of specific nucleic acids in tissues and cells by fluorescence. In addition, sequential FISH, which can identify many RNA transcripts directly in a single cell while maintaining the spatial localization by continuously repeating hybridization and quenching or dehybridization between a primary probe bound to a transcript and a reading probe, is becoming widespread. Sequential FISH methods include MERFISH and seqFISH (sequential fluorescence in situ hybridization).

[0003] In the seqFISH method, the transcripts are sequentially labeled with fluorescent probes by successive rounds of hybridization between the read sequence of the primary probe bound to the transcript and the read probe, and then the barcode consisting of the fluorescent pattern is decoded to uniquely identify the RNA transcripts present in a single cell (see, for example, Patent Documents 1 to 3, etc.). In addition, the seqFISH+ method has also been developed, which can image the mRNAs of 10,000 genes in a single cell with high accuracy and resolution below the diffraction limit using a standard confocal microscope (see, for example, Non-Patent Document 1, etc.). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2016-518843 [Patent Document 2] Patent No. 6605452 [Patent Document 3] International Publication No. 2023 / 282164 [Non-patent literature]

[0005] [Non-Patent Document 1] Eng CH L., et al., “Transcriptome-scale super-resolved imaging in tissues by RNA seqFISH+.”, Nature, Vol. 568, Issue 7751, pp. 235-239, 2019. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a probe design method, a probe design system, and a probe design program that are capable of suppressing false detection. [Means for solving the problem]

[0007] A method for designing a probe according to an embodiment of the present invention includes obtaining a sequence of a molecule to be measured, and generating a sequence unit that satisfies the constraint that a k-mer spanning a linking point of a linked sequence that links the sequence units is not complementary to a k-mer whose occurrence number in the molecule to be measured is equal to or greater than a predetermined value.

[0008] In the above-mentioned method for designing a probe, a sequence unit may be repeated in the linking sequence.

[0009] In the above-mentioned method for designing a probe, sequence units having different sequences may be linked in the linked sequence.

[0010] In the above-mentioned method for designing a probe, at least one of the sequence units having different sequences may be repeated in the linked sequence.

[0011] In the above-mentioned method for designing a probe, a sequence unit may be generated that satisfies the constraint that each k-mer over the entire length of the linked sequence is not complementary to a k-mer that occurs more than a predetermined number of times in the molecule to be measured.

[0012] The above-mentioned method for designing a probe may further include generating k-mers of the molecule to be measured.

[0013] In the above-mentioned method for designing a probe, the length of the k-mer of the measurement target molecule and the length of the k-mer spanning the ligation site may be the same.

[0014] In the above-mentioned method for designing a probe, in generating a sequence unit, a linked sequence consisting of k-mers that is not complementary to k-mers whose number of occurrences of the measurement target molecule is equal to or greater than a predetermined value may be searched for.

[0015] In the above-mentioned method for designing a probe, the sequence of the molecule to be measured may be a base sequence.

[0016] In the above-mentioned method for designing a probe, each of the sequence units having sequences different from each other may bind to a different label, and the above-mentioned method for designing a probe may further include setting the number of repetitions of each of the sequence units having sequences different from each other so that the intensity of the signals emitted by the labels is constant between the different labels.

[0017] In the above-mentioned method for designing a probe, the label may be a fluorescent substance.

[0018] The above-mentioned method for designing a probe may include obtaining a sequence of a molecule to be measured, and generating a sequence unit that satisfies a constraint that a k-mer spanning a linking point of a linked sequence, which is a repeat sequence formed by linking the sequence units, is not complementary to a k-mer whose occurrence number in the molecule to be measured is equal to or greater than a predetermined value.

[0019] The above-mentioned method for designing a probe may include obtaining a sequence of a molecule to be measured, and generating a plurality of sequence units having different sequences from each other, wherein a k-mer spanning a linking point of a linked sequence in which the plurality of sequence units are linked together satisfies a constraint that the k-mer occurring in the molecule to be measured does not become complementary to a k-mer having a number of occurrences equal to or greater than a predetermined value.

[0020] In the above-mentioned method for designing a probe, in the linked sequence, at least any one of a plurality of sequence units may be linked to form a repeat sequence.

[0021] The above-mentioned method for designing a probe may include obtaining a sequence of a molecule to be measured, and generating a plurality of sequence units having different sequences from each other, wherein the k-mer spanning a linking point of a linked sequence, which is a repeat sequence formed by linking each of the plurality of sequence units, satisfies a constraint that the k-mer spanning a linking point of a linked sequence, which links sequence units having different sequences from each other among the plurality of sequence units, satisfies a constraint that the k-mer spanning a linking point of a linked sequence, which links sequence units having different sequences from each other among the plurality of sequence units, does not become complementary to k-mers having a number of occurrences of the molecule to be measured that is a predetermined value or more.

[0022] A probe design system according to an embodiment of the present invention includes a target sequence acquisition unit that acquires the sequence of a target molecule, and a sequence unit generation unit that generates a sequence unit such that a k-mer spanning a linking point of a linked sequence that links the sequence units is not complementary to a k-mer whose occurrence number of target molecules is equal to or greater than a predetermined value.

[0023] In the above-mentioned probe design system, the sequence unit may be repeated in the linking sequence.

[0024] In the above-mentioned probe design system, sequence units having different sequences may be linked in the linked sequence.

[0025] In the above-mentioned system for designing a probe, at least one of the sequence units having different sequences may be repeated in the linked sequence.

[0026] In the above-mentioned probe design system, the sequence unit generation unit may generate a sequence unit that satisfies the constraint that each k-mer over the entire length of the linked sequence is not complementary to a k-mer whose occurrence frequency in the sequence of the molecule to be measured is equal to or greater than a predetermined value.

[0027] The above-mentioned probe design system may further include a partial sequence generator that generates a k-mer of the measurement target molecule.

[0028] In the above-mentioned probe design system, the length of the k-mer of the measurement target molecule and the length of the k-mer spanning the ligation site may be the same.

[0029] In the above-mentioned probe design system, the sequence unit generating section may search for a linked sequence consisting of k-mers that are not complementary to k-mers whose number of occurrences of the measurement target molecule is equal to or greater than a predetermined value.

[0030] In the above-mentioned probe design system, the sequence of the molecule to be measured may be a base sequence.

[0031] In the above-mentioned probe design system, each of the sequence units having sequences different from each other is bound to a different label, and the above-mentioned probe design system may further include a repetition number setting unit that sets the repetition number of each of the sequence units having sequences different from each other so that the intensity of the signals emitted by the labels is constant between the different labels.

[0032] In the above-mentioned probe design system, the label may be a fluorescent substance.

[0033] The above-mentioned probe design system may include a measurement target sequence acquisition unit that acquires the sequence of a measurement target molecule, and a sequence unit generation unit that generates a sequence unit, which satisfies the constraint that a k-mer spanning a linking point of a linked sequence, which is a repeat sequence in which the sequence units are linked, is not complementary to a k-mer whose occurrence number of the measurement target molecule is a predetermined value or more.

[0034] The above-mentioned probe design system may include a target sequence acquisition unit that acquires the sequence of a target molecule, and a sequence unit generation unit that generates a plurality of sequence units having different sequences from each other, the sequence units satisfying the constraint that a k-mer spanning a linking point of a linked sequence in which the plurality of sequence units are linked is not complementary to a k-mer whose occurrence number in the target molecule is a predetermined value or more.

[0035] In the above-mentioned probe design system, a repeat sequence may be formed by linking at least any one of a plurality of sequence units in the linked sequence.

[0036] The above-mentioned probe design system may comprise a measurement target sequence acquisition unit that acquires the sequence of a measurement target molecule, and a sequence unit generation unit that generates a plurality of sequence units having mutually different sequences, the plurality of sequence units being different from each other in which a k-mer spanning a linking point of a linked sequence that is a repeat sequence linking each of the plurality of sequence units satisfies a constraint that the k-mer spanning a linking point of a linked sequence linking sequence units having mutually different sequences among the plurality of sequence units satisfies a constraint that the k-mer spanning a linking point of a linked sequence linking sequence units having mutually different sequences among the plurality of sequence units is not complementary to a k-mer having a predetermined number of occurrences of the measurement target molecule.

[0037] A probe design program according to an embodiment of the present invention may be a probe design program for causing a computer to execute a method including obtaining a sequence of a molecule to be measured, and generating a sequence unit that satisfies the constraint that a k-mer spanning a linking point of a linked sequence that links the sequence units is not complementary to a k-mer whose occurrence number of the molecule to be measured is equal to or greater than a predetermined value.

[0038] In the above-mentioned probe design program, sequence units may be repeated in the linking sequence.

[0039] In the above-mentioned probe design program, sequence units having different sequences may be linked in the linked sequence.

[0040] In the above-mentioned probe design program, at least one of the sequence units having different sequences may be repeated in the linked sequence.

[0041] In the above-mentioned probe design program, a sequence unit may be generated that satisfies the constraint that each k-mer of the entire length of the linked sequence is not complementary to a k-mer whose occurrence number of the measurement target molecule is equal to or exceeds a predetermined value.

[0042] In the above-mentioned probe design program, the method may further include generating k-mers of the measurement target molecule.

[0043] In the above-mentioned probe design program, the length of the k-mer of the measurement target molecule may be the same as the length of the k-mer spanning the ligation site.

[0044] In the above-mentioned probe design program, when generating sequence units, a linked sequence consisting of k-mers that is not complementary to k-mers whose number of occurrences of the measurement target molecule is equal to or greater than a predetermined value may be searched for.

[0045] In the above-mentioned probe design program, the sequence of the molecule to be measured may be a base sequence.

[0046] In the above-mentioned probe design program, each of the sequence units having sequences different from each other may bind to a different label, and the method may further include setting the number of repetitions of each of the sequence units having sequences different from each other so that the intensity of the signal emitted by the label is constant between the different labels.

[0047] In the above-mentioned probe design program, the label may be a fluorescent substance.

[0048] The above-mentioned probe design program may be a probe design program for causing a computer to execute a method including obtaining a sequence of a molecule to be measured, and generating a sequence unit that satisfies the constraint that a k-mer spanning a linking point of a linked sequence, which is a repeat sequence in which the sequence units are linked, is not complementary to a k-mer whose occurrence number in the molecule to be measured is a predetermined value or more.

[0049] The above-mentioned probe design program may be a probe design program for causing a computer to execute a method including obtaining a sequence of a molecule to be measured, and generating a plurality of sequence units having different sequences from each other, wherein a k-mer spanning a linking point of a linked sequence in which the plurality of sequence units are linked satisfies a constraint that the k-mer that spans the linking point of a linked sequence in which the plurality of sequence units are linked is not complementary to a k-mer whose occurrence number of the molecule to be measured is a predetermined value or more.

[0050] In the above-mentioned probe design program, in the linked sequence, at least any one of a plurality of sequence units may be linked to form a repeat sequence.

[0051] The above-mentioned probe design program may be a probe design program for causing a computer to execute a method including: acquiring a sequence of a molecule to be measured; and generating a plurality of sequence units having different sequences from each other, the plurality of sequence units being different from each other in which a k-mer spanning a junction of a linked sequence that is a repeat sequence formed by linking each of the plurality of sequence units satisfies a constraint that the k-mer spanning a junction of a linked sequence that links sequence units having different sequences from each other among the plurality of sequence units satisfies a constraint that the k-mer spanning a junction of a linked sequence that links sequence units having different sequences from each other among the plurality of sequence units is not complementary to a k-mer having a number of occurrences of the molecule to be measured that is a predetermined value or more.

[0052] Probes according to embodiments of the invention comprise one of the sequences shown in SEQ ID NOs: 1 to 16. Probes according to embodiments of the invention consist essentially of one of the sequences shown in SEQ ID NOs: 1 to 16. Probes according to embodiments of the invention consist of one of the sequences shown in SEQ ID NOs: 1 to 16.

[0053] Probes according to embodiments of the invention comprise repeats of one of the sequences shown in SEQ ID NOs: 1 to 16. Probes according to embodiments of the invention consist essentially of repeats of one of the sequences shown in SEQ ID NOs: 1 to 16. Probes according to embodiments of the invention consist of repeats of one of the sequences shown in SEQ ID NOs: 1 to 16.

[0054] Probes according to embodiments of the invention comprise one of the sequences shown in SEQ ID NOs: 17 to 21. Probes according to embodiments of the invention consist essentially of one of the sequences shown in SEQ ID NOs: 17 to 21. Probes according to embodiments of the invention consist of one of the sequences shown in SEQ ID NOs: 17 to 21.

[0055] Probes according to embodiments of the invention comprise a repeat of one of the sequences shown in SEQ ID NOs: 17 to 21. Probes according to embodiments of the invention consist essentially of a repeat of one of the sequences shown in SEQ ID NOs: 17 to 21. Probes according to embodiments of the invention consist of a repeat of one of the sequences shown in SEQ ID NOs: 17 to 21.

[0056] Probes according to embodiments of the invention comprise at least two linked sequences of the sequences shown in SEQ ID NOs: 17 to 21. Probes according to embodiments of the invention consist essentially of at least two linked sequences of the sequences shown in SEQ ID NOs: 17 to 21. Probes according to embodiments of the invention consist of at least two linked sequences of the sequences shown in SEQ ID NOs: 17 to 21. Effect of the Invention

[0057] According to the present invention, it is possible to provide a probe design method, a probe design system, and a probe design program capable of suppressing false detection. [Brief description of the drawings]

[0058] [Figure 1] FIG. 1 is a schematic diagram of a probe according to an embodiment. [Diagram 2] FIG. 2 is a schematic diagram of a probe according to an embodiment. [Diagram 3]FIG. 3 is a schematic diagram of a probe according to an embodiment. [Figure 4] FIG. 4 is a schematic diagram of a probe according to an embodiment. [Diagram 5] FIG. 5 is a schematic diagram of a probe according to an embodiment. [Figure 6] FIG. 6 is a schematic diagram of a probe according to an embodiment. [Figure 7] FIG. 7 is a schematic diagram of a probe according to an embodiment. [Figure 8] FIG. 8 is a schematic diagram of a probe according to an embodiment. [Figure 9] FIG. 9 is a schematic diagram of a probe according to an embodiment. [Figure 10] FIG. 10 is a schematic diagram of a design system for a probe according to the embodiment. [Figure 11] FIG. 11 is a schematic diagram showing a linked sequence consisting of sequence units according to an embodiment, and a partial sequence of the linked sequence. [Figure 12] FIG. 12 is a schematic diagram showing a partial array of an array unit according to an embodiment. [Figure 13] FIG. 13 is a schematic diagram of a probe according to an embodiment. [Figure 14] FIG. 14 is a schematic diagram of a probe according to an embodiment. [Figure 15] FIG. 15 is a schematic diagram of a probe according to an embodiment. [Figure 16] FIG. 16 is a schematic diagram of a probe according to an embodiment. [Figure 17] FIG. 17 is a schematic diagram showing a linked sequence consisting of sequence units according to an embodiment, and a partial sequence of the linked sequence. [Figure 18] FIG. 18 is a schematic diagram showing a partial array of an array unit according to an embodiment. [Figure 19] FIG. 19 is a schematic diagram of a probe according to an embodiment. [Figure 20] FIG. 20 is a schematic diagram of a probe according to an embodiment. [Figure 21]FIG. 21 is a schematic diagram showing a linked sequence made up of sequence units according to an embodiment. [Figure 22] FIG. 22 is a schematic diagram showing a linked sequence made up of sequence units according to an embodiment. [Figure 23] FIG. 23 is a schematic diagram showing a linked sequence made up of sequence units according to an embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0059] The following describes embodiments of the present invention. However, it should not be understood that the following embodiments limit the present invention. From this disclosure, various alternative embodiments, examples, and operating techniques will become apparent to those skilled in the art. It should be understood that the present invention encompasses various embodiments not described herein.

[0060] The measurement target molecule 10 shown in FIG. 1 includes at least one of a detection target molecule 11 and a non-detection target molecule 12. The measurement target molecule 10 may be a biomolecule derived from a human or a non-human animal. The detection target molecule 11 and the non-detection target molecule 12 are, for example, nucleic acids. The nucleic acid may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The DNA contains adenine (A), thymine (T), guanine (G), and cytosine (C) as bases. The RNA contains adenine (A), guanine (G), cytosine (C), and uracil (U) as bases. The RNA may be messenger RNA (mRNA), ribosomal RNA (rRNA), or transfer RNA (tRNA). The nucleic acid has a base sequence. The nucleic acid may also be an artificial nucleic acid. Examples of artificial nucleic acids include bridged nucleic acid (BNA), ethylene-bridged nucleic acid (ENA), peptide nucleic acid (PNA), glycol nucleic acid (GNA), and threose nucleic acid (TNA).

[0061] When detecting the target molecule 11, for example, the target molecule 11 and the primary probe 20 are hybridized. The primary probe 20 includes an antisense sequence 21 that is complementary to at least a part of the sequence of the target molecule 11 and is not complementary to the sequence of the non-target molecule 12, and a reading sequence 22. The reading sequence 22 is connected to the antisense sequence 21. The reading sequence 22 may be connected to the antisense sequence 21 directly or via another compound. The number of reading sequences 22 included in the primary probe 20 is not particularly limited, and may be one or more. The primary probe 20 may include multiple types of reading sequences 22.

[0062] Next, the secondary probe 30 is hybridized to the primary probe 20. The secondary probe 30 includes a sequence 31 complementary to the reading sequence 22A of the primary probe 20, and a label 32. The label 32 may be directly connected to the sequence 31, or may be connected via another compound. The label 32 emits a detectable signal. Examples of the label 32 include fluorescent substances and radioisotopes (RI). The secondary probe 30 binds to the detection target molecule 11 via the primary probe 20, and the primary probe 20 and the secondary probe 30 do not bind to the non-detection target molecule 12, so that the detection target molecule 11 can be specifically detected.

[0063] 2, if the reading sequence 22A of the primary probe 20 is complementary to at least a part of the sequence of the target molecule 11, the secondary probe 30 may not be able to bind to the primary probe 20, and the target molecule 11 may not be detected. Alternatively, if the reading sequence 22B of the primary probe 20 is complementary to at least a part of the sequence of the non-target molecule 12, the secondary probe 30 may bind to the reading sequence 22A of the primary probe 20 hybridized with the non-target molecule 12, and the non-target molecule 12 may be erroneously detected. For this reason, it is preferable that the reading sequence 22 of the primary probe 20 is designed not to be complementary to the sequence of the target molecule 10.

[0064] Furthermore, when the signal emitted by the labels 32 is weak, it may be necessary to increase the number of labels 32 bound to the target molecule 11. In the example shown in Fig. 3, a plurality of primary probes 20A-20D are bound to the target molecule 11, and the same secondary probe 30 is bound to each of the plurality of primary probes 20A-20D, thereby increasing the number of labels 32.

[0065] 4 and 5, one primary probe 20 has multiple reading sequences 22 each having the same sequence, and multiple identical secondary probes 30 are bound to one primary probe 20 to increase the number of labels 32. In these cases, too, it is preferable that the linking sequence consisting of the repeat sequence of the reading sequence 22 of the primary probe 20 is designed so as not to be complementary to the sequence of the molecule 10 to be measured.

[0066] 6, one primary probe 20 has one reading sequence 22, and a secondary probe 36 having a linking sequence consisting of a repeat sequence of the same sequence 31 to which a label 32 is connected is bound to one reading sequence 22 to increase the number of labels 32. In this case, too, it is preferable that the linking sequence consisting of the repeat sequence of sequence 31 of the secondary probe 36 is designed so as not to be complementary to the sequence of the molecule 10 to be measured.

[0067] In addition, it may be required to bind a combination of a plurality of labels 32 that can be distinguished from one another to each of a plurality of detection target molecules 11, and to detect each of the plurality of detection target molecules 11 while distinguishing them from one another. In the example shown in Figs. 7 and 8, one primary probe 20 has a reading sequence 22 and a reading sequence 23 whose sequences are different from each other. A secondary probe 30 having a sequence 31 complementary to the reading sequence 22 and a label 32 is bound to the reading sequence 22. A secondary probe 33 having a sequence 34 complementary to the reading sequence 23 and a label 35 that emits a signal different from that of the label 32 is bound to the reading sequence 23. In these cases, too, it is preferable that the linking sequence consisting of the reading sequence 22 and the reading sequence 23 of the primary probe 20 is designed not to be complementary to the sequence of the measurement target molecule 10.

[0068] 9, one primary probe 20 has one reading sequence 22, and one reading sequence 22 is bound to a secondary probe 37 having a linking sequence consisting of sequence 31 to which label 32 is connected and sequence 34 different from sequence 31 to which label 35 that emits a signal different from that of label 32 is connected. In this case as well, it is preferable that the linking sequence consisting of sequence 31 and sequence 34 of secondary probe 37 is designed not to be complementary to the sequence of the molecule 10 to be measured.

[0069] 10, the probe design system according to the embodiment includes a measurement target sequence acquisition unit 301 that acquires a sequence of a measurement target molecule, and a sequence unit generation unit 302 that generates a sequence unit that satisfies a constraint that a k-mer spanning a linking point of a linked sequence in which the sequence units are linked is not complementary to a k-mer whose occurrence number in the measurement target molecule is equal to or greater than a predetermined value. The measurement target sequence acquisition unit 301 and the sequence unit generation unit 302 are included in a computer 300, for example.

[0070] The sequence of the measurement target molecule acquired by the measurement target sequence acquisition unit 301 is the sequence of the measurement target molecule that may be contained in a sample to be measured using the designed probe. As described above, the measurement target molecule includes at least one of a detection target molecule and a non-detection target molecule. The detection target molecule and the non-detection target molecule are, for example, nucleic acids, which may be DNA or RNA, and which may be mRNA, rRNA, or tRNA. The nucleic acid may also be an artificial nucleic acid.

[0071] The method by which the measurement target sequence acquisition unit 301 acquires the sequence of the measurement target molecule is not particularly limited. An operator may input the sequence of the measurement target molecule to the measurement target sequence acquisition unit 301 from the input device 351. Alternatively, the measurement target sequence acquisition unit 301 may acquire the sequence of the measurement target molecule from a database 352 that stores the sequence of the measurement target molecule. The input device 351 and the database 352 are electrically connected to the measurement target sequence acquisition unit 301. The input device 351 and the database 352 may be connected to the measurement target sequence acquisition unit 301 via a network line.

[0072] The probe design system according to the embodiment may further include a partial sequence generating unit 303 that generates k-mers of the sequence of the target molecule. The partial sequence generating unit 303 generates k-mers of length k, where k is a natural number, by shifting the length by one base from one end of the sequence of the target molecule, for example. The base length of the k-mer of the target molecule is, for example, 3 or more, 4 or more, 5 or more, or 10 or more. The base length of the k-mer of the target molecule is, for example, 14 or less. The partial sequence generating unit 303 may calculate the number of occurrences of each of a plurality of k-mers in the sequence of the target molecule that has been generated. The number of occurrences of the k-mer is also called the k-mer count.

[0073] The sequence unit generated by the sequence unit generating section 302 is used, for example, as the read sequence of the primary probe or the sequence of the secondary probe described above. The base length of the sequence unit is not particularly limited, but may be, for example, 15 or more, 20 or more, 30 or more, or 40 or more. For example, when generating a sequence unit constituting a repeat sequence, the sequence unit generating section 302 generates a sequence unit 40 as shown in FIG. 11, which is a sequence unit 40, and satisfies the constraint that all k-mers 42A, 42B, 42C, etc. of a linked sequence 41 formed by linking two identical sequence units 40 are not complementary to k-mers whose number of occurrences of the sequence of the measurement target molecule is equal to or greater than a predetermined value. It is possible that a plurality of sequence units 40 that satisfy the constraint may be generated.

[0074] The predetermined value of the number of occurrences of the k-mer of the molecule to be measured is, for example, 1, 2, 3, 4, 5, or 10. The smaller the predetermined value of the number of occurrences, the lower the probability that the linked sequence 41 hybridizes with the sequence of the molecule to be measured. However, the smaller the predetermined value of the number of occurrences, the more difficult it may be to generate a sequence unit 40 that satisfies the constraints. In that case, the predetermined value of the number of occurrences may be increased until a sequence unit 40 that satisfies the constraints can be generated. In addition, when multiple sequence units 40 that satisfy the constraints are generated, the predetermined value of the number of occurrences may be decreased. Alternatively, the sequence units 40 may be sorted in order of decreasing the predetermined value of the number of occurrences. The same applies below.

[0075] When linking the sequence units 40, the sequence units 40 are linked directly to each other, and no other bases are inserted between the linked sequence units 40. The same applies below. The base length of the k-mer of the sequence of the measurement target molecule and the base length of the k-mer of the linked sequence 41 may be the same. The same applies below. The base length of each of the multiple k-mers of the linked sequence 41 is, for example, 3 or more, 4 or more, 5 or more, or 10 or more, and 14 or less.

[0076] For example, the sequence unit generating unit 302 generates one sequence unit that satisfies the above constraints by searching for a linked sequence 41 in which two identical sequence units are linked and which is composed of k-mers that are not complementary to k-mers in the sequence of the target molecule having a k-mer count of a predetermined value or more. Here, if a linked sequence 41 composed of k-mers that are not complementary to k-mers in the sequence of the target molecule having a k-mer count of 1 or more is searched for, the k-mer spanning the linking point of the linked sequence 41 will not be completely complementary to the k-mer in the sequence of the target molecule.

[0077] The number of k-mers that are not complementary to k-mers with a k-mer count of the sequence of the target molecule that is equal to or greater than a certain value may be limited. However, because of the constraint that the same sequence units must be linked together, the same k-mer can be used multiple times. Therefore, it is possible to reduce the consumption of resources for k-mers that satisfy the constraint.

[0078] Furthermore, even if the generated sequence unit 40 forms a repeat sequence in which the sequence unit is repeated three or more times, the k-mer spanning the junction of the repeat sequence will not be complementary to a k-mer whose occurrence in the measured molecule is equal to or greater than a predetermined value.

[0079] As described above, in order to increase the signal intensity of the label bound to the primary probe, the primary probe may contain a repeat sequence in which a plurality of identical sequence units are linked as a read sequence, or the secondary probe may contain a repeat sequence in which a plurality of identical sequence units are linked. Here, even if a single sequence unit 40 is generated that satisfies the constraint that all k-mers 42A, 42B, 42C, etc. are not complementary to multiple k-mers in the sequence of the target molecule as shown in Figure 12, when the sequence units 40 are linked to each other, it cannot be guaranteed that the k-mers 42K and 42L that span the linking portion of the two sequence units 40 shown in Figure 11 will not be complementary to the k-mer of the target molecule.

[0080] In contrast, by using a sequence unit generated by the probe design system of the embodiment, even if the primary probe or secondary probe contains a repeat sequence in which multiple identical sequence units are linked, hybridization of the repeat sequence with the sequence of the molecule to be measured is suppressed.

[0081] In the primary probe 20 shown in Figure 13(a), the same reading sequence 22 is repeated twice at both ends of the antisense sequence 21. In the primary probe 20 shown in Figure 13(b), the same reading sequence 22 is repeated twice at one end of the antisense sequence 21, and the same reading sequence 22 is repeated three times at the other end of the antisense sequence 21.

[0082] In the primary probe 20 shown in Figure 13(c), the same reading sequence 22 is repeated three times at both ends of the antisense sequence 21. In the primary probe 20 shown in Figure 13(d), the same reading sequence 22 is repeated three times at one end of the antisense sequence 21, and the same reading sequence 22 is repeated four times at the other end of the antisense sequence 21.

[0083] If a sequence unit generated by the probe design system according to the embodiment is used as the read sequence 22 shown in FIG. 13, hybridization of the repeat sequence of the read sequence 22 with the sequence of the molecule to be measured is suppressed.

[0084] In the primary probe 20 shown in Fig. 14(a), one reading sequence 22 is connected to each end of the antisense sequence 21, and a secondary probe 36 having a linking sequence consisting of a repeat sequence of the same sequence 31 to which a label 32 is connected is bound to the reading sequence 22. If the sequence unit generated by the probe design system according to the embodiment is used as the sequence 31 of the secondary probe 36 shown in Fig. 14(a), the repeat sequence of the sequence 31 of the secondary probe 36 is suppressed from hybridizing with the sequence of the molecule to be measured.

[0085] In the primary probe 20 shown in Fig. 14(b), repeat sequences of the same reading sequence 22 are connected to both ends of the antisense sequence 21. In addition, a secondary probe 36 having a linking sequence consisting of a repeat sequence of the same sequence 31 to which a label 32 is connected is bound to each of the reading sequences 22. If two sequence units that satisfy the constraints generated by the probe design system according to the embodiment are used as the reading sequence 22 and the sequence 31 of the secondary probe 36 shown in Fig. 14(b), respectively, hybridization of the repeat sequence of the reading sequence 22 and the repeat sequence of the sequence 31 of the secondary probe 36 with the sequence of the molecule to be measured is suppressed.

[0086] In the primary probe 20 shown in Figure 15(a), the reading sequence 22 is repeated twice at one end of the antisense sequence 21, and the reading sequence 23, which has a different sequence from the reading sequence 22, is repeated twice at the other end of the antisense sequence 21. In the primary probe 20 shown in Figure 15(b), the reading sequence 22 is repeated twice at one end of the antisense sequence 21, and the reading sequence 23, which has a different sequence from the reading sequence 22, is repeated three times at the other end of the antisense sequence 21.

[0087] In the primary probe 20 shown in Figure 15(c), the reading sequence 22 is repeated three times at one end of the antisense sequence 21, and the reading sequence 23, which has a different sequence from the reading sequence 22, is repeated three times at the other end of the antisense sequence 21. In the primary probe 20 shown in Figure 15(d), the reading sequence 22 is repeated three times at one end of the antisense sequence 21, and the reading sequence 23, which has a different sequence from the reading sequence 22, is repeated four times at the other end of the antisense sequence 21.

[0088] 15, a secondary probe 30 having an array 31 to which a label 32 is connected is bound to a reading sequence 22. A secondary probe 33 having an array 34 to which a label 35 different from the label 32 is connected is bound to a reading sequence 23. If two sequence units satisfying the constraints generated by the probe design system according to the embodiment are used as the reading sequence 22 and the reading sequence 23 shown in FIG. 15, hybridization of the repeat sequence of the reading sequence 22 and the repeat sequence of the reading sequence 23 with the sequence of the molecule to be measured is suppressed.

[0089] 16(a), a single read sequence 22 is connected to one end of an antisense sequence 21, and a read sequence 23 having a sequence different from that of the read sequence 22 is connected to the other end of the antisense sequence 21. A secondary probe 36 having a linking sequence consisting of a repeat sequence of the same sequence 31 to which a label 32 is connected is bound to the read sequence 22. A secondary probe 39 having a linking sequence consisting of a repeat sequence of the same sequence 34 to which a label 35 different from the label 32 is connected is bound to the read sequence 23.

[0090] By using two sequence units that satisfy the constraints generated by the probe design system of the embodiment as sequence 31 of secondary probe 36 and sequence 34 of secondary probe 39 shown in Figure 16(a), respectively, hybridization of the repeat sequence of sequence 31 of secondary probe 36 and the repeat sequence of sequence 34 of secondary probe 39 with the sequence of the molecule to be measured is suppressed.

[0091] 16(b), a repeat sequence of a reading sequence 22 is connected to one end of an antisense sequence 21, and a repeat sequence of a reading sequence 23, which is different from the reading sequence 22, is connected to the other end of the antisense sequence 21. A secondary probe 36 having a linking sequence consisting of a repeat sequence of a sequence 31 to which a label 32 is connected is bound to each of the reading sequences 22. A secondary probe 39 having a linking sequence consisting of a repeat sequence of a sequence 34 to which a label 35 different from the label 32 is connected is bound to each of the reading sequences 23.

[0092] If four sequence units that satisfy the constraints generated by the probe design system of the embodiment are used as the reading sequence 22, the reading sequence 23, the sequence 31 of the secondary probe 36, and the sequence 34 of the secondary probe 39 shown in Figure 16(b), hybridization of the repeat sequence of the reading sequence 22, the repeat sequence of the reading sequence 23, the repeat sequence of the sequence 31 of the secondary probe 36, and the repeat sequence of the sequence 34 of the secondary probe 39 with the sequence of the molecule to be measured is suppressed.

[0093] When generating a plurality of sequence units having different sequences, the sequence unit generating section 302 generates a plurality of sequence units 50A, 50B, which are different in sequence from each other, and which satisfy a constraint that all k-mers 52A, 52B, 52C, etc. of a linked sequence 51 formed by linking the plurality of sequence units 50A, 50B are not complementary to k-mers whose number of occurrences of the sequence of the measurement target molecule is equal to or greater than a predetermined value, as shown in Fig. 17. The constraint may include the order in which the plurality of sequence units having different sequences are linked in the continuous sequence.

[0094] For example, the sequence unit generating unit 302 generates a plurality of sequence units having mutually different sequences that satisfy the above-mentioned constraints by searching for a linked sequence 51 in which a plurality of sequence units having mutually different sequences are linked, the linked sequence 51 being composed of k-mers that are not complementary to k-mers in the sequence of the measurement target molecule having a k-mer count of a predetermined value or more. Here, if a linked sequence 51 composed of k-mers that are not complementary to k-mers in the sequence of the measurement target molecule having a k-mer count of 1 or more is searched for, the k-mer spanning the linking point of the linked sequence 51 will not be completely complementary to the k-mer in the sequence of the measurement target molecule.

[0095] As described above, in order to increase the number of types of labels bound to the primary probe, the primary probe may contain a sequence in which multiple sequence units with different sequences are linked as a read sequence, or the secondary probe may contain a sequence in which multiple sequence units with different sequences are linked. Here, as shown in Figure 18, even if a single sequence unit 50A, which is a single sequence unit, and which satisfies the constraint that all k-mers 52A, 52B, 52C... are not complementary to multiple k-mers in the sequence of the target molecule, and a single sequence unit 50B, which is a single sequence unit, and which satisfies the constraint that all k-mers 52M, 52N, 52O... are not complementary to multiple k-mers in the sequence of the target molecule, are separately generated, when the sequence units 50A and 50B are linked, it cannot be guaranteed that the k-mers 52K and 52L that span the linking portion of the two sequence units 50A and 50B shown in Figure 17 will not be complementary to the k-mer of the target molecule.

[0096] In contrast, by using the sequence unit generated by the probe design system of the embodiment, even if the primary probe or secondary probe contains a sequence in which multiple sequence units with different sequences are linked together, hybridization of the linked sequence with the sequence of the molecule to be measured is suppressed.

[0097] In the primary probe 20 shown in Fig. 19(a), a reading sequence 22 and a reading sequence 23 having a sequence different from that of the reading sequence 22 are linked to one end of an antisense sequence 21. In the primary probe 20 shown in Fig. 19(b), a reading sequence 22, a reading sequence 23, and a reading sequence 22 are linked to one end of an antisense sequence 21.

[0098] In the primary probe 20 shown in Figure 19(c), a reading sequence 22, a reading sequence 23, a reading sequence 22, and a reading sequence 23 are linked to one end of an antisense sequence 21. In the primary probe 20 shown in Figure 19(d), a reading sequence 22, a reading sequence 23, a reading sequence 22, a reading sequence 23, and a reading sequence 22 are linked to one end of an antisense sequence 21.

[0099] 19, a secondary probe 30 having a sequence 31 complementary to the reading sequence 22 and a label 32 is bound to the reading sequence 22. A secondary probe 33 having a sequence 34 complementary to the reading sequence 23 and a label 35 different from the label 32 is bound to the reading sequence 23. If two sequence units having different sequences generated by the probe design system according to the embodiment are used as the reading sequence 22 and the reading sequence 23 shown in FIG. 19, hybridization of the linked sequence consisting of the reading sequence 22 and the reading sequence 23 of the primary probe 20 with the sequence of the molecule to be measured is suppressed.

[0100] 20(a), a reading sequence 22 and a reading sequence 23, the sequence of which is different from that of the reading sequence 22, are linked to one end of an antisense sequence 21. In addition, a reading sequence 24, the sequence of which is different from that of the reading sequence 22 and the reading sequence 23, and a reading sequence 25, the sequence of which is different from that of the reading sequence 22, the reading sequence 23, and the reading sequence 24, are linked to the other end of the antisense sequence 21.

[0101] In the primary probe 20 shown in Fig. 20(b), a reading sequence 22, a reading sequence 23, a reading sequence 22, and a reading sequence 23 are linked to one end of an antisense sequence 21. In addition, a reading sequence 24, a reading sequence 25, a reading sequence 24, and a reading sequence 25 are linked to the other end of the antisense sequence 21.

[0102] 20, a secondary probe 30 having a sequence 31 complementary to the reading sequence 22 and a label 32 is bound to the reading sequence 22. A secondary probe 33 having a sequence 34 complementary to the reading sequence 23 and a label 35 different from the label 32 is bound to the reading sequence 23. A secondary probe 130 having a sequence 131 complementary to the reading sequence 24 and a label 132 different from the labels 32 and 35 is bound to the reading sequence 24. A secondary probe 133 having a sequence 134 complementary to the reading sequence 25 and a label 135 different from the labels 32, 35 and 132 is bound to the reading sequence 25.

[0103] By using one of two pairs of sequence units having different sequences generated by the probe design system of the embodiment as the reading sequence 22 and the reading sequence 23 shown in Figure 20 and using the other pair as the reading sequence 24 and the reading sequence 25, hybridization of the linked sequence consisting of the reading sequence 22 and the reading sequence 23 of the primary probe 20 and the linked sequence consisting of the reading sequence 24 and the reading sequence 25 with the sequence of the molecule to be measured is suppressed.

[0104] 10 may generate a plurality of sequence units having different sequences, the plurality of sequence units being different from each other in which k-mers across the linking points of a linked sequence, which is a repeat sequence obtained by linking each of the plurality of sequence units, satisfy a constraint that k-mers across the linking points of a linked sequence obtained by linking sequence units having different sequences among the plurality of sequence units are not complementary to k-mers having a predetermined number of occurrences of the measurement target molecule or more, and also satisfy a constraint that k-mers across the linking points of a linked sequence obtained by linking sequence units having different sequences among the plurality of sequence units are not complementary to k-mers having a predetermined number of occurrences of the measurement target molecule or more. The constraint may include the order in which the sequence units having different sequences are linked in the linked sequence obtained by linking sequence units having different sequences.

[0105] For example, when first, second, and third sequence units having different sequences are generated by this method, the repeat sequence of the first sequence unit has a k-mer spanning the junction of the repeat sequence that is not complementary to a k-mer in which the number of occurrences of the measurement target molecule is equal to or greater than a predetermined value, as shown in Figure 21. The number of repetitions p of the first sequence unit in the repeat sequence of the first sequence unit is arbitrary.

[0106] In addition, the repeat sequence of the second sequence unit is such that the k-mer spanning the junction of the repeat sequence is not complementary to the k-mer in which the number of occurrences of the measurement target molecule is equal to or greater than a predetermined value. The number of repetitions q of the second sequence unit in the repeat sequence of the second sequence unit is arbitrary.

[0107] In addition, the repeat sequence of the third sequence unit is such that the k-mer spanning the junction of the repeat sequence is not complementary to the k-mer in which the number of occurrences of the measurement target molecule is equal to or greater than a predetermined value. The number of repetitions r of the third sequence unit in the repeat sequence of the third sequence unit is arbitrary.

[0108] Furthermore, when the first, second, and third sequence units having mutually different sequences are generated by the above method, the k-mer across the linking point of the linked sequence of the first sequence unit and the second sequence unit is not complementary to the k-mer having a predetermined number of occurrences of the measurement target molecule or more, as shown in Fig. 22. Also, the k-mer across the linking point of the linked sequence of the second sequence unit and the third sequence unit is not complementary to the k-mer having a predetermined number of occurrences of the measurement target molecule or more.

[0109] In addition, when the first, second, and third sequence units having different sequences are generated by the above method, the k-mer across the linking point of the linking sequence of the repeat sequence of the first sequence unit having an arbitrary repeat number p, the repeat sequence of the second sequence unit having an arbitrary repeat number q, and the repeat sequence of the third sequence unit having an arbitrary repeat number r is not complementary to the k-mer in which the number of occurrences of the measurement target molecule is equal to or greater than a predetermined value, as shown in Figure 23. p, q, and r may be natural numbers or positive integers including 0.

[0110] The linking sequence shown in FIG. 23 can be used as, for example, a read sequence for a primary probe, and can also be used as a sequence for binding a label for a secondary probe. In the linking sequence shown in FIG. 23, different labels can be bound to the first, second, and third sequence units, respectively. Therefore, in the example of the linking sequence shown in FIG. 23, three types of labels can be bound. Furthermore, since the number of repetitions can be set for each repeat sequence, it is possible to set the number of labels bound to the linking sequence for each type of label. Specifically, the linking sequence shown in FIG. 23 can bind p first labels, q second labels, and r third labels. Therefore, by setting the number of repetitions for each of the first, second, and third sequence units, the signal intensity for each label can be set arbitrarily.

[0111] As shown in Fig. 10, the probe design system according to the embodiment may further include a repeat number setting unit 304 for setting the number of repeats of sequence units in a repeat sequence formed by linking a plurality of sequence units having different sequences, so that the intensity of signals emitted by the labels is constant between different labels. In the example shown in Fig. 23, the signal intensity of the first label is S1, the signal intensity of the second label is S2, and the signal intensity of the third label is S3, and the number of repeats p of the first sequence unit, the number of repeats q of the second sequence unit, and the number of repeats r of the third sequence unit may be set so that S1 x p, S2 x q, and S3 x r are equal.

[0112] Conventionally, the Hybrid Chain Reaction (HCR) method is used as a fluorescence amplification technique. However, in the HCR method, the fluorescent probes continue to be linked as long as the reaction continues, so the fluorescence intensity cannot be set. In addition, the HCR method cannot use a plurality of different fluorescent substances. In contrast, in the method according to the embodiment, it is possible to set the number of bonds for each of a plurality of labels such as fluorescent substances.

[0113] Example 1 The entire genome sequence of human IMR-90 cells was obtained. In addition, k-mers of the obtained genome sequence were generated. Next, MANE v1.0 was used to search for repeat sequences in which 15-base sequence units were linked and which were composed of k-mers that were not complementary to k-mers with a k-mer count of 5 or more in the genome sequence, to generate multiple sequence units. Furthermore, from the multiple sequence units, the base sequences shown in SEQ ID NOs: 1 to 16 in Table 1, which do not have forward complementary sequences and reverse complementary sequences of 8 bases or more each other, were obtained. Each of the base sequences shown in SEQ ID NOs: 1 to 16 does not substantially hybridize with the genome sequence even when it forms a repeat sequence. [Table 1]

[0114] Example 2 The whole genome sequence of human IMR-90 cells was obtained. In addition, k-mers of the obtained genome sequence were generated. Next, using MANE v1.0, the base sequences shown in SEQ ID NOs: 17 to 21 in Table 2 were obtained, which are five 16-base sequence units with different sequences, and the k-mers of the repeat sequence obtained by linking each of these five sequence units satisfy the constraint that they are not complementary to k-mers with a k-mer count of 5 or more in the genome sequence, and the k-mers of the linked sequence obtained by linking five sequence units with different sequences in order satisfy the constraint that they are not complementary to k-mers with a k-mer count of 5 or more in the genome sequence.

[0115] Each of the base sequences shown in SEQ ID NOs: 17 to 21 does not substantially hybridize with a genome sequence even if it forms a repeat sequence. A linked sequence in which the base sequences shown in SEQ ID NOs: 17 to 21 are linked in order does not substantially hybridize with a genome sequence. A linked sequence in which the repeat sequences of each of the base sequences shown in SEQ ID NOs: 17 to 21 are linked in order does not substantially hybridize with a genome sequence. [Table 2] [Explanation of symbols]

[0116] 10... Molecule to be measured, 11... Molecule to be detected, 12... Non-target molecule, 20... Primary probe, 21... Antisense sequence, 22... Read sequence, 23... Read sequence, 24... Read sequence, 25... Read sequence, 30... Secondary probe, 31... Sequence, 32... Label, 33... Secondary probe, 34... Sequence, 35... Label, 36... Secondary probe, 37... Secondary probe, 39... Secondary probe, 4 0...sequence unit, 41...linked sequence, 50...sequence unit, 51...linked sequence, 130...secondary probe, 131...sequence, 132...label, 133...secondary probe, 134...sequence, 135...label, 300...computer, 301...measurement target sequence acquisition section, 302...sequence unit generation section, 303...partial sequence generation section, 304...repetition number setting section, 351...input device, 352...database,

Claims

1. Obtaining a sequence of a molecule to be measured; generating a sequence unit, the sequence unit satisfying a constraint that a k-mer spanning a linking portion of a linking sequence formed by linking the sequence unit is not complementary to a k-mer in which the number of occurrences of the measurement target molecule is equal to or greater than a predetermined value; A method for designing a probe comprising:

2. The method for designing a probe according to claim 1 , wherein the sequence unit is repeated in the linked sequence.

3. The method for designing a probe according to claim 1 , wherein the sequence units linked in the linked sequence have sequences different from each other.

4. The method for designing a probe according to claim 3 , wherein at least one of the sequence units having mutually different sequences is repeated in the linked sequence.

5. The method for designing a probe according to claim 1, wherein the sequence unit is generated such that each k-mer of the entire length of the linked sequence satisfies a constraint that the number of occurrences of the measurement target molecule is not a predetermined value or more and is not complementary to the k-mer.

6. Each of the sequence units, which are different from each other in sequence, is bound to a different label; and determining a repeat number of each of the sequence units having different sequences so that the intensity of the signals emitted by the different labels is constant. A method for designing a probe according to claim 1.

7. a measurement target sequence acquisition unit that acquires a sequence of a measurement target molecule; a sequence unit generating unit that generates a sequence unit, the sequence unit satisfying a constraint that a k-mer spanning a linking portion of a linked sequence in which the sequence units are linked is not complementary to a k-mer in which the number of occurrences of the measurement target molecule is equal to or greater than a predetermined value; A probe design system comprising:

8. The probe design system according to claim 7 , wherein the sequence unit is repeated in the linking sequence.

9. The system for designing a probe according to claim 7 , wherein the sequence units having different sequences are linked in the linked sequence.

10. The system for designing a probe according to claim 9 , wherein at least one sequence unit among the sequence units whose sequences differ from each other is repeated in the linked sequence.

11. The probe design system according to claim 7, wherein the sequence unit generation unit generates the sequence unit such that each k-mer of the entire length of the linked sequence satisfies a constraint that the number of occurrences of the target molecule is not equal to or greater than a predetermined value and is not complementary to any k-mer of the target molecule.

12. Each of the sequence units, which are different from each other in sequence, is bound to a different label; a repetition number setting unit that sets the repetition number of each of the arrangement units having different arrangements so that the intensity of the signals emitted by the different markers is constant; The probe design system according to claim 7.

13. On the computer, Obtaining a sequence of a molecule to be measured; generating a sequence unit, the sequence unit satisfying a constraint that a k-mer spanning a linking portion of a linking sequence formed by linking the sequence unit is not complementary to a k-mer in which the number of occurrences of the measurement target molecule is equal to or greater than a predetermined value; A probe design program for carrying out the method comprising:

14. The probe design program according to claim 13 , wherein the sequence unit is repeated in the linked sequence.

15. The probe design program according to claim 13 , wherein the sequence units having sequences different from each other are linked in the linked sequence.

16. The probe design program according to claim 15 , wherein at least one of the sequence units having mutually different sequences is repeated in the linked sequence.

17. The probe design program according to claim 13, which generates the sequence unit such that each k-mer of the full length of the linked sequence satisfies a constraint that the number of occurrences of the measurement target molecule is not complementary to a k-mer having a predetermined value or more.

18. Each of the sequence units, which are different from each other in sequence, is bound to a different label; The method further includes setting a number of repetitions of each of the sequence units having different sequences such that the intensity of the signals emitted by the different labels is constant. A program for designing the probe according to claim 13.

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