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

Abstract

This system for designing a probe comprises: a measurement target sequence acquisition section 301 for acquiring the sequence of a molecule being 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 molecule being measured is greater than or equal to a predetermined value.

Description

PROBE DESIGN METHOD, PROBE DESIGN SYSTEM, AND PROBE DESIGN PROGRAM

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

[0002] In recent years, transcriptome analysis has become increasingly important in understanding the relationship between changes in gene expression 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 using fluorescence. Furthermore, in FISH, sequential FISH, which continuously repeats hybridization and quenching or dehybridization between a primary probe bound to a transcript and a reading probe, can directly identify many RNA transcripts within a single cell while maintaining their spatial localization, has become widespread. Sequential FISH methods include MERFSH and seqFISH (sequential fluorescence in situ hybridization).

[0003] In the seqFISH method, transcripts are sequentially labeled with fluorescent probes through successive rounds of hybridization between the readout sequences of primary probes bound to the transcripts and the readout probes, and then barcodes consisting of fluorescent patterns are decoded to uniquely identify RNA transcripts present in a single cell (see, for example, Patent Documents 1 to 3, etc.). Also, a seqFISH+ method has been developed that can image the mRNAs of 10,000 genes in a single cell with high precision and sub-diffraction-limit resolution using a standard confocal microscope (see, for example, Non-Patent Document 1, etc.).

[0004] JP 2016-518843 A, Patent No. 6605452 A, International Publication No. 2023 / 282164

[0005] Eng CH L., et al., “Transcriptome-scale super-resolved imaging in tissues by RNA seqFISH+.”, Nature, Vol. 568, Issue 7751, pp. 235-239, 2019.

[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.

[0007] A method for designing a probe according to an aspect of the present invention includes obtaining the 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 formed by linking the sequence units is not complementary to a k-mer in which the number of occurrences of the molecule to be measured is equal to or greater than a predetermined value.

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

[0009] In the above-mentioned method for designing a probe, the linked sequence may include sequence units that are different from each other in 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-described method for designing a probe, 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 in which the number of occurrences of the molecule to be measured is equal to or greater than a predetermined value.

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

[0013] In the above-described method for designing a probe, the length of the k-mer of the molecule to be measured may be the same as the length of the k-mer spanning the linkage site.

[0014] In the above-described method for designing a probe, when generating a sequence unit, a linked sequence consisting of k-mers that is not complementary to k-mers in which the number of occurrences of the molecule to be measured 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 probe design method, each of the sequence units having different sequences may bind to a different label, and the above-mentioned probe design method may further include setting the number of repetitions of each of the sequence units having different sequences 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 in which the number of occurrences of 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, which satisfy a constraint that a k-mer spanning a linking point of a linked sequence formed by linking the plurality of sequence units is not complementary to a k-mer in which the number of occurrences of the molecule to be measured is equal to or greater than a predetermined value.

[0020] In the above-mentioned method for designing a probe, the linked sequence may comprise a repeat sequence formed by linking at least any one of a plurality of sequence units.

[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 whose sequences are different from each other, wherein the k-mer spanning a linkage point 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 linkage point of a linked sequence formed by linking sequence units that are different from each other among the plurality of sequence units satisfies a constraint that the k-mer spanning a linkage point of a linked sequence formed by linking sequence units that are different from each other in sequences is not complementary to a k-mer in which the number of occurrences of the molecule to be measured is equal to or greater than a predetermined value.

[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 that satisfies the constraint that a k-mer spanning a linking point in a linked sequence formed by linking the sequence units is not complementary to a k-mer in which the number of occurrences of the target molecule 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-described probe design system, sequence units having different sequences may be linked in the linked sequence.

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

[0026] In the above-described probe design system, the sequence unit generation unit may generate sequence units that satisfy the constraint that each k-mer over the entire length of the linked sequence is not complementary to any k-mer in the sequence of the molecule to be measured that appears more than a predetermined number of times.

[0027] The above-mentioned probe design system may further comprise a partial sequence generation unit that generates a k-mer of the molecule to be measured.

[0028] In the above-described probe design system, the length of the k-mer of the molecule to be measured may be the same as the length of the k-mer spanning the ligation site.

[0029] In the above-described 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 in which the number of occurrences of the measurement target molecule is equal to or greater than a predetermined value.

[0030] In the above-described 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 different sequences may bind 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 different sequences so that the intensity of the signal emitted by the label is constant between the different labels.

[0032] In the above-described 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 that satisfies the 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 in which the number of occurrences of the measurement target molecule is equal to or greater than a predetermined value.

[0034] 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 plurality of sequence units having mutually different sequences, wherein the plurality of sequence units are linked together and a k-mer spanning a linking point of a linked sequence satisfies a constraint that the k-mer that spans the linking point of a measurement target molecule appears more than a predetermined number of times.

[0035] In the above-described 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 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 plurality of sequence units that are different from each other in sequence, wherein the plurality of sequence units are different from each other in sequence, and the k-mer spanning the junction of a linked sequence that is a repeat sequence formed by linking each of the plurality of sequence units satisfies the constraint that the k-mer spanning the junction of a linked sequence formed by linking sequence units that are different from each other in sequence among the plurality of sequence units 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.

[0037] A probe design program according to an aspect of the present invention may be a probe design program for causing a computer to execute a method including: acquiring the 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 in which the sequence units are linked is not complementary to a k-mer in which the number of occurrences of the molecule to be measured is equal to or greater than a predetermined value.

[0038] In the above-mentioned probe design program, the sequence unit may be repeated in the linked 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 in which the number of occurrences of the target molecule is equal to or greater than a predetermined value.

[0042] In the above-mentioned probe design program, the method may further include generating k-mers of the molecules to be measured.

[0043] In the above-mentioned probe design program, the length of the k-mer of the molecule to be measured may be the same as the length of the k-mer spanning the linkage 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 in which the number of occurrences of the molecule to be measured 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 different sequences may bind to a different label, and the method may further include setting the number of repetitions of each of the sequence units having different sequences 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 probe design program may be a probe design program for causing a computer to execute a method including: acquiring the 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 in which the number of occurrences of the molecule to be measured is equal to or greater than a predetermined value.

[0049] The probe design program may be a probe design program for causing a computer to execute a method including: acquiring the sequence of a molecule to be measured; and generating a plurality of sequence units having different sequences from one another, wherein a k-mer spanning a linking point of a linked sequence formed by linking the plurality of sequence units satisfies a constraint that the k-mer that spans the linking point of a molecule to be measured does not become complementary to a k-mer in which the number of occurrences of the molecule to be measured is equal to or greater than a predetermined value.

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

[0051] The probe design program may be a program for designing a probe that causes a computer to execute a method including: acquiring the sequence of a molecule to be measured; and generating a plurality of sequence units whose sequences are different from each other, wherein the k-mer spanning the junction of a linked sequence that is a repeat sequence formed by linking each of the plurality of sequence units satisfies the constraint that the k-mer spanning the junction of a linked sequence formed by linking sequence units that are different from each other among the plurality of sequence units satisfies the constraint that the k-mer spanning the junction of a linked sequence formed by linking sequence units that are different from each other in sequences is not complementary to the k-mer in which the number of times the molecule to be measured appears is equal to or greater than a predetermined value.

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

[0053] Probes according to aspects of the invention comprise a repeat sequence of one of the sequences shown in SEQ ID NOs: 1 to 16. Probes according to aspects of the invention consist essentially of a repeat sequence of one of the sequences shown in SEQ ID NOs: 1 to 16. Probes according to aspects of the invention consist of a repeat sequence of one of the sequences shown in SEQ ID NOs: 1 to 16.

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

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

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

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

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

[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 target molecule 10 shown in FIG. 1 includes at least one of a target molecule 11 and a non-target molecule 12. The target molecule 10 may be a biomolecule derived from a human or a non-human animal. The target molecule 11 and the non-target molecule 12 are, for example, nucleic acids. The nucleic acid may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). DNA contains adenine (A), thymine (T), guanine (G), and cytosine (C) as bases. 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 acids (BNA), ethylene-bridged nucleic acids (ENA), peptide nucleic acids (PNA), glycol nucleic acids (GNA), and threose nucleic acids (TNA).

[0061] When detecting the target molecule 11, for example, the target molecule 11 is hybridized with the primary probe 20. The primary probe 20 comprises an antisense sequence 21 that is complementary to at least a portion 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 provided in the primary probe 20 is not particularly limited and may be one or more. The primary probe 20 may comprise multiple types of reading sequences 22.

[0062] Next, the secondary probe 30 is hybridized to the primary probe 20. The secondary probe 30 comprises 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 target molecule 11 via the primary probe 20, while the primary probe 20 and secondary probe 30 do not bind to the non-target molecule 12, making it possible to specifically detect the target molecule 11.

[0063] 2, if the reading sequence 22A of the primary probe 20 is complementary to at least a portion 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 portion 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 that has hybridized with the non-target molecule 12, and the non-target molecule 12 may be erroneously detected. Therefore, it is preferable that the reading sequence 22 of the primary probe 20 is designed so as not to be complementary to the sequence of the target molecule 10.

[0064] Furthermore, if 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 Figure 3, multiple primary probes 20A-20D are bound to the target molecule 11, and the same secondary probe 30 is bound to each of the multiple primary probes 20A-20D, thereby increasing the number of labels 32.

[0065] 4 and 5, one primary probe 20 comprises a plurality of reading sequences 22 each having the same sequence, and a plurality of identical secondary probes 30 are bound to one primary probe 20 to increase the number of labels 32. In these cases as well, 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 one reading sequence 22 is bound to 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, thereby increasing the number of labels 32. In this case, too, it is preferable that the linking sequence consisting of a 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] Furthermore, it may be necessary to bind a combination of a plurality of mutually distinguishable labels 32 to each of a plurality of target molecules 11, thereby enabling the detection of each of the plurality of target molecules 11 while distinguishing between them. In the examples shown in Figures 7 and 8, one primary probe 20 comprises a reading sequence 22 and a reading sequence 23, which have different sequences. A secondary probe 30 comprising a sequence 31 complementary to the reading sequence 22 and a label 32 is bound to the reading sequence 22. A secondary probe 33 comprising a sequence 34 complementary to the reading sequence 23 and a label 35 that emits a signal different from the label 32 is bound to the reading sequence 23. In these cases as well, 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 so as not to be complementary to the sequence of the target molecule 10.

[0068] 9, one primary probe 20 has one reading sequence 22, and a secondary probe 37 is bound to one reading sequence 22, the secondary probe 37 having a linking sequence consisting of sequence 31 to which label 32 is attached and sequence 34 different from sequence 31 to which label 35 that emits a signal different from label 32 is attached. 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 so as 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 the sequence of a measurement target molecule, and a sequence unit generation unit 302 that generates a sequence unit that satisfies the constraint that a k-mer spanning a linkage point of a linked sequence formed by linking the sequence units 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. 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 using 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 generation unit 303 that generates k-mers of the sequence of the target molecule. The partial sequence generation unit 303 generates k-mers of length k bases, where k is a natural number, by shifting the bases one by one 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 generation unit 303 may calculate the number of occurrences of each of the multiple k-mers in the generated sequence of the target molecule. The number of occurrences of the k-mer is also referred to as the k-mer count.

[0073] The sequence units generated by the sequence unit generation unit 302 are 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 sequence units constituting a repeat sequence, the sequence unit generation unit 302 generates a sequence unit 40, as shown in FIG. 11 , that 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 occurrence count in the sequence of the molecule to be measured is equal to or greater than a predetermined value. It is possible that multiple sequence units 40 that satisfy the constraint may be generated.

[0074] The predetermined value for 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 for the number of occurrences, the lower the probability that the linked sequence 41 will hybridize with the sequence of the molecule to be measured. However, the smaller the predetermined value for 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 for the number of occurrences may be increased until a sequence unit 40 that satisfies the constraints can be generated. Furthermore, if multiple sequence units 40 that satisfy the constraints have been generated, the predetermined value for the number of occurrences may be decreased. Alternatively, the sequence units 40 may be sorted in order of decreasing the predetermined value for the number of occurrences. The same applies hereinafter.

[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 hereinafter. The base length of the k-mer of the sequence of the molecule to be measured and the base length of the k-mer of the linked sequence 41 may be the same. The same applies hereinafter. 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 generation 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 together and which is composed of k-mers that are not complementary to k-mers in the sequence of the molecule to be measured whose k-mer count is 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 molecule to be measured whose k-mer count is 1 or more is searched for, the k-mer spanning the linking point in the linked sequence 41 will not be completely complementary to the k-mer in the sequence of the molecule to be measured.

[0077] There may be a limit to the number of k-mers that are not complementary to k-mers with a k-mer count equal to or greater than a predetermined value in the sequence of the molecule being measured. However, because of the constraint that the same sequence units must be linked together, the same k-mer can be used multiple times. This makes it 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 linkage point of the repeat sequence will not be complementary to the k-mer in which the number of occurrences of the molecule to be measured 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, a repeat sequence formed by linking multiple identical sequence units may be included in the primary probe as a readout sequence, or a repeat sequence formed by linking multiple identical sequence units may be included in the secondary probe. Even if a single sequence unit 40 is generated that satisfies the constraint that all k-mers 42A, 42B, 42C, ... 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 together, it cannot be guaranteed that the k-mers 42K and 42L spanning 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 the sequence units 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 the sequence unit generated by the probe design system according to the embodiment is used as the read sequence 22 shown in FIG. 13, the repeat sequence of the read sequence 22 is prevented from hybridizing with the sequence of the molecule to be measured.

[0084] In the primary probe 20 shown in Figure 14(a), one reading sequence 22 is connected to each end of the antisense sequence 21, and a secondary probe 36 is bound to the reading sequence 22, which includes a linking sequence consisting of a repeat sequence of the same sequence 31 connected to a label 32. 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 Figure 14(a), hybridization of the repeat sequence of the sequence 31 of the secondary probe 36 with the sequence of the molecule to be measured is suppressed.

[0085] In the primary probe 20 shown in Figure 14(b), repeat sequences of the same reading sequence 22 are connected to both ends of the antisense sequence 21. Furthermore, each reading sequence 22 is bound to a secondary probe 36 having a linking sequence consisting of a repeat sequence of the same sequence 31 connected to a label 32. 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 Figure 14(b), 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 sequence different from that of 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 sequence different from that of 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 sequence different from that of 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 sequence different from that of the reading sequence 22, is repeated four times at the other end of the antisense sequence 21.

[0088] 15, a secondary probe 30 having a sequence 31 connected to a label 32 is bound to a reading sequence 22. A secondary probe 33 having a sequence 34 having a label 35 connected to a label different from the label 32 is bound to a reading sequence 23. 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 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, which has 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 is bound to the read sequence 22, and includes a linking sequence consisting of a repeat sequence of the same sequence 31 to which a label 32 is connected. A secondary probe 39 is bound to the read sequence 23, and includes 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.

[0090] If two sequence units that satisfy the constraints generated by the probe design system of the embodiment are used as sequence 31 of secondary probe 36 and sequence 34 of secondary probe 39 shown in Figure 16 (a), 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. Furthermore, 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 reading sequence 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 reading sequence 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] Furthermore, when generating a plurality of sequence units having mutually different sequences, the sequence unit generating unit 302 generates a plurality of sequence units 50A, 50B, as shown in Fig. 17, in which the plurality of sequence units 50A, 50B have mutually different sequences, and satisfy the 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 in the sequence of the molecule to be measured is equal to or greater than a predetermined value. The constraint may include the order in which the plurality of sequence units having mutually different sequences are linked in the contiguous sequence.

[0094] For example, the sequence unit generation unit 302 generates a plurality of sequence units with mutually different sequences that satisfy the above constraints by searching for a linked sequence 51 in which a plurality of sequence units with 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 molecule to be measured, whose k-mer count is 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 molecule to be measured, whose k-mer count is 1 or more, is searched for, the k-mer spanning the linking point in the linked sequence 51 will not be completely complementary to the k-mer in the sequence of the molecule to be measured.

[0095] As described above, in order to increase the number of labels that can be bound to a primary probe, a primary probe may contain a readout sequence in which a plurality of sequence units having different sequences are linked, or a secondary probe may contain a sequence in which a plurality of sequence units having different sequences are linked. Here, as shown in Figure 18 , even if a single sequence unit 50A that 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 that 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-mers of the target molecule.

[0096] In contrast, by using the sequence units 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 Figure 19(a), a reading sequence 22 and a reading sequence 23, which has a sequence different from that of the reading sequence 22, are linked to one end of the antisense sequence 21. In the primary probe 20 shown in Figure 19(b), a reading sequence 22, a reading sequence 23, and a reading sequence 22 are linked to one end of the 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 the 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 the antisense sequence 21.

[0099] 19, a secondary probe 30 comprising a sequence 31 complementary to the reading sequence 22 and a label 32 is bound to the reading sequence 22. A secondary probe 33 comprising 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, which has a different sequence from the reading sequence 22, are linked to one end of an antisense sequence 21. In addition, a reading sequence 24, which has a different sequence from the reading sequences 22 and 23, and a reading sequence 25, which has a different sequence from the reading sequences 22, 23, and 24, are linked to the other end of the antisense sequence 21.

[0101] In the primary probe 20 shown in Figure 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 comprising a sequence 31 complementary to the reading sequence 22 and a label 32 binds to the reading sequence 22. A secondary probe 33 comprising a sequence 34 complementary to the reading sequence 23 and a label 35 different from the label 32 binds to the reading sequence 23. A secondary probe 130 comprising a sequence 131 complementary to the reading sequence 24 and a label 132 different from the labels 32 and 35 binds to the reading sequence 24. A secondary probe 133 comprising a sequence 134 complementary to the reading sequence 25 and a label 135 different from the labels 32, 35, and 132 binds to the reading sequence 25.

[0103] If one of the two sets of two sequence units having different sequences generated by the probe design system of the embodiment is used as the reading sequence 22 and the reading sequence 23 shown in Figure 20, and the other set is used 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 having different sequences satisfying a constraint that a 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, is not complementary to a k-mer in which the number of occurrences of the molecule to be measured is a predetermined value or more, and the plurality of sequence units having different sequences, the k-mer spanning a linking point of a linked sequence formed by linking the sequence units having different sequences, is not complementary to a k-mer in which the number of occurrences of the molecule to be measured is a predetermined value or more. The constraint may include the order in which the sequence units having different sequences are linked in the linked sequence formed by linking the 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 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] Furthermore, 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 molecule to be measured 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] Furthermore, 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 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 first, second, and third sequence units having mutually different sequences are generated by the above method, the k-mer spanning the linking point of the linked sequence of the first sequence unit and the second sequence unit is not complementary to the k-mer in which the number of molecules to be measured appears to be a predetermined value or more, as shown in Figure 22. Furthermore, the k-mer spanning the linking point of the linked sequence of the second sequence unit and the third sequence unit is not complementary to the k-mer in which the number of molecules to be measured appears to be a predetermined value or more.

[0109] In addition, when first, second, and third sequence units having mutually different sequences are generated by the above method, as shown in Figure 23, in a linked sequence of a repeat sequence of a first sequence unit having an arbitrary repeat number p, a repeat sequence of a second sequence unit having an arbitrary repeat number q, and a repeat sequence of a third sequence unit having an arbitrary repeat number r, the k-mer spanning the linking point of the linked sequence 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. p, q, and r may be natural numbers or positive integers including 0.

[0110] The linking sequence shown in FIG. 23 can be used, for example, as a read sequence for a primary probe, or as a sequence for binding labels to a secondary probe. In the linking sequence shown in FIG. 23, different labels can be bound to the first, second, and third sequence units. Therefore, in the example 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 to be 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 as desired.

[0111] 10 , the probe design system according to the embodiment may further include a repeat number setting unit 304 that sets the number of repeats of a sequence unit in a repeat sequence formed by linking a plurality of sequence units having different sequences so that the intensity of signals emitted by the different labels is constant. 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×p, S2×q, and S3×r are equal.

[0112] Conventionally, a fluorescence amplification technique is the Hybrid Chain Reaction (HCR) method. However, in the HCR method, fluorescent probes remain linked as long as the reaction continues, making it impossible to set the fluorescence intensity. Furthermore, the HCR method does not allow the use of multiple different fluorescent substances. In contrast, the method according to the embodiment makes it possible to set the number of bonds for each of multiple labels, such as fluorescent substances.

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

[0114] Example 2 The entire genome sequence of human IMR-90 cells was obtained. Furthermore, k-mers were generated from the obtained genome sequence. Next, using MANE v1.0, the base sequences shown in SEQ ID NOS: 17 to 21 in Table 2 were obtained, which consisted of five 16-base sequence units that differ from one another, and which satisfied the constraint that k-mers of repeat sequences formed by linking each of these five sequence units were not complementary to k-mers with a k-mer count of 5 or more in the genome sequence, and which also satisfied the constraint that k-mers of linked sequences formed by linking five sequence units that differ from one another in order were 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 when 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 the base sequences shown in SEQ ID NOs: 17 to 21 are linked in order does not substantially hybridize with a genome sequence.

[0116] 10... Molecule to be measured, 11... Molecule to be detected, 12... Molecule not to be detected, 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, 40... 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 unit, 302... Sequence unit generation unit, 303... Partial sequence generation unit, 304... Repetition number setting unit, 351... Input device, 352... Database

Claims

1. A method for designing a probe comprising: acquiring 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 in which the sequence unit is linked is not complementary to a k-mer in which the number of occurrences of the molecule to be measured is equal to or greater than a predetermined value.

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 having different sequences are linked in the linked sequence.

4. The method for designing a probe according to claim 3, wherein at least one of the sequence units having 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 full length of the linked sequence satisfies the constraint that the number of occurrences of the target molecule is not complementary to a k-mer that is equal to or greater than a predetermined value.

6. The method for designing a probe according to claim 1, further comprising: setting the number of repetitions of each of the sequence units having mutually different sequences so that each of the sequence units having mutually different sequences binds to a different label, and the intensity of the signal emitted by the label is constant between the different labels.

7. A probe design system comprising: 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, the sequence unit satisfying the 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 in which the number of occurrences of the measurement target molecule is equal to or greater than a predetermined value.

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 of the sequence units having different sequences is repeated in the linked sequence.

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

12. The probe design system of claim 7, further comprising a repetition number setting unit that sets the number of repetitions of each of the sequence units of the sequence units that differ from each other in sequence so that each of the sequence units that differ from each other in sequence binds to a different label and the intensity of the signal emitted by the label is constant between the different labels.

13. A probe design program for causing a computer to execute a method comprising: 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 in which the sequence units are linked is not complementary to a k-mer in which the number of occurrences of the molecule to be measured is equal to or greater than a predetermined value.

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 different sequences are linked in the linked sequence.

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

17. A 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 the constraint that the number of occurrences of the target molecule is not complementary to a k-mer that is equal to or greater than a predetermined value.

18. The probe design program of claim 13, wherein each of the sequence units having different sequences binds to a different label, and the method further comprises setting the number of repetitions of each of the sequence units having different sequences so that the intensity of the signal emitted by the label is constant between the different labels.

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